RNA-guided retargeting of Sleeping Beauty transposition in human cells

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Abstract

An ideal tool for gene therapy would enable efficient gene integration at predetermined sites in the human genome. Here we demonstrate biased genome-wide integration of the Sleeping Beauty (SB) transposon by combining it with components of the CRISPR/Cas9 system. We provide proof-of-concept that it is possible to influence the target site selection of SB by fusing it to a catalytically inactive Cas9 (dCas9) and by providing a single guide RNA (sgRNA) against the human Alu retrotransposon. Enrichment of transposon integrations was dependent on the sgRNA, and occurred in an asymmetric pattern with a bias towards sites in a relatively narrow, 300-bp window downstream of the sgRNA targets. Our data indicate that the targeting mechanism specified by CRISPR/Cas9 forces integration into genomic regions that are otherwise poor targets for SB transposition. Future modifications of this technology may allow the development of methods for specific gene insertion for precision genetic engineering.

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DNA sequence data generated and analysed during this study are included in the manuscript and Source Data Files 1-4.

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Adrian Kovač

Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany

Competing interests
No competing interests declared.
Csaba Miskey

Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany

Competing interests
No competing interests declared.
Michael Menzel

Bioinformatics, University of Applied Sciences, Giessen, Germany

Competing interests
No competing interests declared.
Esther Grueso

Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany

Competing interests
No competing interests declared.
Andreas Gogol-Döring

Bioinformatics, University of Applied Sciences, Giessen, Germany

Competing interests
No competing interests declared.
Zoltan Ivics

Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany

For correspondence
zoltan.ivics@pei.de

Competing interests
Zoltan Ivics, Patent applications around targeted transposon integration technology(Patent Nos. EP1594971B1, EP1594972B1 and EP1594973B1)..

"This ORCID iD identifies the author of this article:" 0000-0002-7803-6658

Funding

No external funding was received for this work.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.